Use of derivatized nanoparticles to minimize growth of micro-organisms in hot filled drinks

ABSTRACT

A method and article for removing a selected metal-ion from a solution. The method included providing a container for holding a liquid, the container having an internal surface having a metal-ion sequestering agent and antimicrobial agent for inhibiting growth of microbes in the liquid, filling the container with the liquid in an open environment, closing the container with the liquid contained therein, and shipping the container for use of the liquid without any or reduced further processing of the container containing the liquid.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. patent application Ser. No.10/823,443 filed Apr. 13, 2004 entitled USE OF DERIVATIZED NANOPARTICLESTO MINIMIZE GROWTH OF MICRO-ORGANISMS IN HOT FILLED DRINKS by Richard W.Wien.

Reference is made to commonly assigned U.S. patent application Ser. No.______ filed herewith entitled ARTICLE FOR INHIBITING MICROBIAL GROWTHby Joseph F. Bringley, David L. Patton, Richard W. Wien, Yannick J. F.Lerat (docket 87834), U.S. patent application Ser. No. ______ filedherewith entitled CONTAINER FOR INHIBITING MICROBIAL GROWTH IN LIQUIDNUTRIENTS by David L. Patton, Joseph F. Bringley, Richard W. Wien, JohnM. Pochan, Yannick J. F. Lerat (docket 87472); U.S. patent applicationSer. No. ______ filed herewith entitled ARTICLE FOR INHIBITING MICROBIALGROWTH IN PHYSIOLOGICAL FLUIDS by Joseph F. Bringley, David L. Patton,Richard W. Wien, Yannick J. F. Lerat (docket 87833) the disclosures ofwhich are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to using metal-ion sequestering agents ina container filling process for removing bio-essential designatedmetal-ions from a liquid nutrient for inhibiting growth of microbes inthe liquid nutrient.

BACKGROUND OF THE INVENTION

During the process of filling containers with certain beverages andfoodstuffs, air borne micro-organisms may enter the containers after theflash pasteurization or pasteurization part of the process. Thesemicro-organisms such as yeast, spores, bacteria, etc. will grow in thenutrient rich beverage or food, ruining the taste or even causinghazardous micro-biological contamination. While some beverages arepackaged by aseptic means or by utilizing preservatives, many otherbeverages for example fruit juices, teas and isotonic drinks are“hot-filled”. “Hot-filling” involves the filling of a container with aliquid beverage having some elevated temperature (typically, at about180-200° F.). The container is capped and allowed to cool, producing avacuum therein. The process of hot filling of beverages and foods isused to kill micro-organisms that enter the container during the fillingof the beverage or food containers. Hot filling requires containers bemade of certain materials or constructed in a certain fashion such asthicker walls to withstand the hot filling process. The energy requiredfor hot filling adds to the cost of the filling process. Temperaturesrequired for hot filling have a detrimental effect on the flavor of thebeverage. Other methods of filling, such as aseptic filling, requirelarge capital expenditures and maintenance of class 5 clean roomconditions.

It has been recognized that small concentrations of metal-ions play animportant role in biological processes. For example, Mn, Fe, Ca, Zn, Cuand Al are essential bio-metals, and are required for most, if not all,living systems. Metal-ions play a crucial role in oxygen transport inliving systems, and regulate the function of genes and replication inmany cellular systems. Calcium is an important structural element in theformation of bones and other hard tissues. Mn, Cu and Fe are involved inmetabolism and enzymatic processes. At high concentrations, metals maybecome toxic to living systems and the organism may experience diseaseor illness if the level cannot be controlled. As a result, theavailability and concentrations of metal-ions in biological environmentsis a major factor in determining the abundance, growth-rate and healthof plant, animal and micro-organism populations. It has been recognizedthat iron is an essential biological element, and that all livingorganisms require iron for survival and replication. Although, theoccurrence and concentration of iron is relatively high on the earth'ssurface, the availability of “free” iron is severely limited by theextreme insolubility of iron in aqueous environments. As a result, manyorganisms have developed complex methods of procuring “free” iron forsurvival and replication.

Methods for packaging drinks and liquid foodstuffs are needed that areable to improve food quality, to increase shelf-life, to protect frommicrobial contamination, and to do so in a manner that is safe andenvironmentally clean. Methods are needed that are able to target andremove specific, biologically important, metal-ions while leaving intactthe concentrations of beneficial metal-ions.

Problem To be Solved by the Invention

“Hot filling” provides various advantages over aseptic or preservativepackaging, among them lower capital and operational cost (over asepticsystems), and the elimination of the need for preservatives (the heat ofthe beverage has a sanitizing effect). The hot headspace in the filledbottle also reduces the carrying capacity of oxygen therein, limitingoxidation of the contents. There is however a problem in the hot fillingof beverages and foods when used to kill air borne micro-organisms thatenter the containers during the filling process after the flashpasteurization or pasteurization of the beverage or food. Hot fillingrequires containers be made of certain materials or constructed in acertain fashion such as the use of thicker walls, more material, andspecific shapes to withstand the hot filling process. The energyrequired for hot filling adds to the cost of the filling process.Temperatures required for hot filling have a detrimental effect on theflavor of the beverage. Hot filling adds additional time to themanufacturing process in both the heating and cooling of the containers.The manufacturers of the beverages and foodstuffs are loathe to addantimicrobial materials directly to the beverages and foods becausethese may potentially alter the color or taste of items such asbeverages and foodstuffs, and in the worst case may be harmful to thepersons using or consuming those items. The wide spread use ofantimicrobial materials may cause further problems in that disposal ofthe items containing these materials cannot be accomplished withoutimpacting the biological health of the landfill or other site ofdisposal, and further the antimicrobial compounds may leach intosurrounding rivers, lakes and water supplies. The wide spread use ofantimicrobial materials may cause yet further problems in thatmicro-organisms may develop resistance to these materials and newinfectious microbes and new diseases may develop.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there isprovided a method of removing a selected metal-ion from a solution,comprising the steps of:

-   -   a. providing a container for holding a liquid, the container        having an internal surface having a metal-ion sequestering agent        provided on at least a portion of the internal surface for        removing designated metal-ions from the liquid and an        antimicrobial agent for reducing and/or maintaining the amount        of microbes in said liquid to a prescribed condition;    -   b. filling the container with the liquid in an open environment;    -   c. closing the container with the liquid contained therein; and    -   d. shipping the container for use of the liquid without any        further processing of the container containing the liquid.

In accordance with another aspect of the present invention, there isprovided a method for bottling a liquid having a pH equal to or greaterthan about 2.5, comprising the steps of:

-   -   a. providing a container having a metal-ion sequestering agent        and an antimicrobial agent provided on at least a portion of the        internal surface for inhibiting growth of microbes;    -   b. filling the container with a liquid having a pH equal to or        greater than about 2.5;    -   c. closing the container with the liquid contained therein; and    -   d. shipping the container for use without any further        sterilization of the liquid and/or container.

In accordance with still another aspect of the present invention, thereis provided an article for inhibiting the growth of microbes in a liquidnutrient when placed in contact with the liquid nutrient, the articlehaving a metal-ion sequestering agent and an antimicrobial agent suchthat when the article is placed in contact with the liquid nutrient themetal-ion sequestering agent and the antimicrobial agent inhibits thegrowth of microbes in the liquid nutrient.

These and other aspects, objects, features and advantages of the presentinvention will be more clearly understood and appreciated from a reviewof the following detailed description of the preferred embodiments andappended claims and by reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the detailed description of the preferred embodiments of theinvention presented below, reference is made to the accompanyingdrawings in which:

FIG. 1 is a schematic of a hot fill bottling process made in accordancewith the prior art;

FIG. 2 illustrates a cross section of a fluid container made inaccordance with the prior art;

FIG. 3 is an enlarged partial cross sectional view of a portion of thecontainer of FIG. 2;

FIG. 4 is a view similar to FIG. 3 illustrating a container made inaccordance with the present invention;

FIG. 5 illustrates a modified bottle and cap assembly also made inaccordance with the present invention;

FIG. 6 is a schematic top plan view of the bottle and cap of FIG. 5;

FIG. 7 is an enlarged partial cross sectional view of the bottle and captaken along line 7-7 of FIG. 6;

FIG. 8 is a schematic view of another embodiment of the presentinvention illustrating one method for applying a coating to the interiorsurface of a bottle made in accordance with the present invention;

FIG. 9 is an enlarged partial cross sectional view of a portion of thebottle of FIG. 8 illustrating the sprayed coating of the metal-ionsequestering agent; and

FIG. 10 is a schematic of a hot fill bottling process made in accordancewith the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Referring to FIG. 1, there is illustrated a schematic view of a priorart system of a “hot fill” process 5 for bottling certain types ofliquid nutrients 8 such as isotonic beverages having a pH equal to orgreater than about 2.5 made in accordance with the prior art. Drinkssuch as Gatorade™ or PowerAide™, fruit drinks, and teas are examples ofisotonic beverages. The bottling process typically begins with cleanedand sanitized containers such as bottles 12 formed from glass or using apolymer as described in FIGS. 2 and 3. The “hot fill” process of FIG. 1may also be used for filling various other containers, for example butnot limited to, bags, stand up pouches, juice boxes, cans, etc. Afterformulation, the beverage 8 is usually stored in a tank 10 until it ispumped via a pump 15 through a pasteurizer 20 to a filler station 25.Excess beverage may be pumped back to the tank 10 via line 26. Althoughthese systems may integrate one or more functions, such systems aretypically exposed in one way or another to the environment such thatcontaminants or other micro-organisms can enter into the filling orbottling process at one or more locations along the processing path 27.At the same time sanitized bottles 12 are also supplied to the fillerstation 25 wherein the beverage 8 is dispensed into the bottle 12. Thebottle 12 is then moved to a capper 35 where the bottle 12 is sealed.Afterward the filled sealed bottle 12 is transported through a heatingtunnel 40 where the beverage in the sealed bottle 12 is heated to atemperature typically about 180-200° F. The bottle 12 is thentransported through a cooling tunnel 45 where it is inverted to insurethe entire inside of the bottle 12 is subjected to the heated beveragebefore it is discharged to the packaging station 50, packaged andsubsequently shipped at the shipping station 55.

Referring to FIG. 2, there is illustrated a cross-sectional view of atypical prior art container 12. The container 12 comprises the bottle 12holding the liquid nutrient 8, for example the isotonic beverage. Thecontainer 12 may be made of one or more layers of a plastic polymerusing various molding processes known by those skilled in the art.Examples of polymers used in the manufacture of plastic bottles are PET(polyethylene terephthalate), PP (polypropylene), LDPE (low densitypolyethylene) and HDPE (high density polyethylene). FIG. 3 illustrates aplastic bottle 12 formed using two different polymeric layers 60 and 65.However it is to be understood that the container 12 may comprise anydesired number of layers.

The term inhibition of microbial-growth, or a material which “inhibits”microbial growth, is used by the authors to mean materials which eitherprevent microbial growth, or subsequently kills microbes so that thepopulation is within acceptable limits, or materials which significantlyretard the growth processes of microbes or maintain the level ormicrobes to a prescribed level or range. The prescribed level may varywidely depending upon the microbe and its pathogenicity. Generally it ispreferred that harmful organisms are present at no more than 10organisms/ml and preferably less than 1 organism/ml.

Antimicrobial agents which kill microbes or substantially reduce thepopulation of microbes are often referred to as biocidal agents, whilematerials which simply slow or retard normal biological growth arereferred to as biostatic agents. The preferred impact upon the microbialpopulation may vary widely depending upon the application. Forpathogenic organisms (such as E. coli O157:H7) a biocidal effect is morepreferred, while for less harmful organisms, a biostatic impact may bepreferred. Generally, it is preferred that microbiological organismsremain at a level which is not harmful to the consumer or user of thatparticular article

A fluid container, such a container 12 illustrated in FIG. 4 anddiscussed in greater detail later herein, made in accordance with thepresent invention is especially useful for containing a liquid nutrient,for example a beverage, having a pH equal to or greater than about 2.5.The higher the pH, the more beneficial is obtained from a container madein accordance with the present invention. Thus, if the pH is 3.0 or 4.0or greater the present invention will provide greater benefit. Thecontainer is designed to have an interior surface having a metal-ionsequestering agent for removing a designated metal-ion from a liquidnutrient for inhibiting growth of microbes in said liquid nutrient. Itis preferred that the metal-ion sequestering agent is immobilized on thesupport structure and has a high-selectivity for biologically importantmetal-ions such as Mn, Zn, Cu and Fe. This is important becausemetal-ion sequestrants that are not immobilized may diffuse through thematerial or polymeric layers of the container and dissolve into thecontents of the beverage. Metal-ions complexed by dissolved sequestrantswill not be sequestered within the surfaces of the container but may beavailable for use by micro-organisms.

It is preferred that the fluid container made in accordance with thepresent invention comprises a polymer containing said metal-ionsequestrant. The container may comprise the polymer itself containingsaid metal-ion sequestrant, or alternatively, the metal-ion sequestrantmay be contained with a polymeric layer attached to a support structure.It is preferred that said polymer is permeable to water. It is importantthat the polymer is permeable to water because permeability facilitatesthe contact of the target metal-ions with the metal-ion sequestrant,which, in turn, facilitates the sequestration of the metal-ions withinthe polymer or polymeric layer. A measure of the permeability of variouspolymeric addenda to water is given by the permeability coefficient, Pthat is given byP=(quantity of permeate)(film thickness)/[area×time×(pressure dropacross the film)]

Permeability coefficients and diffusion data of water for variouspolymers are discussed by J. Comyn, in Polymer Permeability, Elsevier,N.Y., 1985 and in “Permeability and Other Film Properties Of Plasticsand Elastomers”, Plastics Design Library, NY, 1995. The higher thepermeability coefficient, the greater the water permeability of thepolymeric media. The permeability coefficient of a particular polymermay vary depending upon the density, crystallinity, molecular weight,degree of cross-linking, and the presence of addenda such ascoating-aids, plasticizers, etc. It is preferred that the polymer has awater permeability of greater than 1000 [(cm³ cm)/(cm²sec/Pa)]×10¹³. Itis further preferred that the polymer has a water permeability ofgreater than 5000 [(cm³ cm)/(cm²sec/Pa)]×10¹³. Preferred polymers forpractice of the invention are polyvinyl alcohol, cellophane, water-basedpolyurethanes, polyester, nylon, high nitrile resins,polyethylene-polyvinyl alcohol copolymer, polystyrene, ethyl cellulose,cellulose acetate, cellulose nitrate, aqueous latexes, polyacrylic acid,polystyrene sulfonate, polyamide, polymethacrylate, polyethyleneterephthalate, polystyrene, polyethylene, polypropylene orpolyacrylonitrile. It is preferred that the metal-ion sequestrantcomprises 0.1 to 50.0% by weight of the polymer, and more preferably 1%to 10% by weight of the polymer.

In a preferred embodiment, the container 12 comprises a plurality oflayers having an outer layer having a metal-ion sequestering agent. Inanother preferred embodiment, the container comprises a plurality oflayers comprising a barrier layer for contact with said beverage orfoodstuff and an inner layer having said sequestering agent, said innerlayer having a first side adjacent said barrier layer, and said barrierlayer allowing liquid to pass through to said inner layer. Multiplelayers may be necessary to provide a rigid structure able to containfoodstuffs and to provide physical robustness. In a particular casethere may be provided a second outer layer on the second side of saidinner layer. It is preferred that both the first and second outer layercomprise a barrier layer that allows liquid to pass through to saidinner layer. The barrier layer does not contain the metal-ionsequestrant. However, the primary purpose of the barrier layer is toprovide a barrier through which micro-organisms cannot pass. It isimportant to limit or eliminate, the direct contact of micro-organismswith the metal-ion sequestrant or the layer containing the metal-ionsequestrant, since many micro-organisms, under conditions of irondeficiency, may bio-synthesize molecules which are strong chelators foriron and other metals. These bio-synthetic molecules are called“siderophores” and their primary purpose is to procure iron for themicro-organisms. Thus, if the micro-organisms are allowed to directlycontact the metal-ion sequestrant they may find a rich source of ironthere and begin to colonize directly at these surfaces. The siderophoresproduced by the micro-organisms may compete with the metal-ionsequestrant for the iron (or other bio-essential metal) at theirsurfaces. The barrier layer of the invention does not contain themetal-ion sequestrant, and because micro-organisms are large, they maynot pass or diffuse through the barrier layer. The barrier layer thusprevents contact of the micro-organisms with the polymeric layercontaining the metal-ion sequestrant of the invention.

It is preferred that the metal-ion sequestrant has a high-affinity forbiologically important metal-ions such as Mn, Zn, Cu and Fe. A measureof the “affinity” of metal-ion sequestrants for various metal-ions isgiven by the stability constant (also often referred to as criticalstability constants, complex formation constants, equilibrium constants,or formation constants) of that sequestrant for a given metal-ion.Stability constants are discussed at length in “Critical StabilityConstants”, A. E. Martell and R. M. Smith, Vols. 1-4, Plenum, NY (1977),“Inorganic Chemistry in Biology and Medicine”, Chapter 17, ACS SymposiumSeries, Washington, D.C. (1980), and by R. D. Hancock and A. E. Martell,Chem. Rev. vol. 89, p. 1875-1914 (1989). The ability of a specificmolecule or ligand to sequester a metal-ion may depend also upon the pH,the concentrations of interfering ions, and the rate of complexformation (kinetics). Generally, however, the greater the stabilityconstant the greater the binding affinity for that particular metal-ion.Often the stability constants are expressed as the natural logarithm ofthe stability constant. Herein the stability constant for the reactionof a metal-ion (M) and a sequestrant or ligand (L) is defined asfollows:M+nL⇄ML_(n)

where the stability constant is β_(n)=[ML_(n)]/[M][L]^(n), wherein[ML_(n)] is the concentration of “complexed” metal-ion, [M] is theconcentration of free (uncomplexed) metal-ion and [L] is theconcentration of free ligand. The log of the stability constant is logβ_(n), and n is the number of ligands which coordinate with the metal.It follows from the above equation that if β_(n) is very large, theconcentration of “free” metal-ion will be very low. Ligands with a highstability constant (or affinity) generally have a stability constantgreater than 10¹⁰ or a log stability constant greater than 10 for thetarget metal. Preferably the ligands have a stability constant greaterthan 10¹⁵ for the target metal-ion. Table 1 lists common ligands (orsequestrants) and the natural logarithm of their stability constants(log β_(n)) for selected metal-ions. TABLE 1 Common ligands (orsequestrants) and the natural logarithm of their stability constants(log β_(n)) for selected metal-ions. Ligand Ca Mg Cu(II) Fe(III) Al AgZn alpha-amino carboxylates EDTA 10.6 8.8 18.7 25.1 7.2 16.4 DTPA 10.89.3 21.4 28.0 18.7 8.1 15.1 CDTA 13.2 21.9 30.0 NTA 24.3 DPTA 6.7 5.317.2 20.1 18.7 5.3 PDTA 7.3 18.8 15.2 citric Acid 3.50 3.37 5.9 11.57.98 9.9 salicylic acid 35.3 Hydroxamates Desferroxamine B 30.6acetohydroxamic 28 acid Catechols 1,8-dihydroxy 37 naphthalene 3,6sulfonic acid MECAMS 44 4-LICAMS 27.4 3,4-LICAMS 16.2 438-hydroxyquinoline 36.9 disulfocatechol 5.8 6.9 14.3 20.4 16.6

EDTA is ethylenediamine tetra acetic acid and salts thereof, DTPA isdiethylenetriaminepentaacetic acid and salts thereof, DPTA isHydroxylpropylenediaminetetraacetic acid and salts thereof, NTA isnitrilotriacetic acid and salts thereof, CDTA is 1,2-cyclohexanediaminetetra acetic acid and salts thereof, PDTA is propylenediammine tetraacetic acid and salts thereof. Desferroxamine B is a commerciallyavailable iron chelating drug, desferal®. MECAMS, 4-LICAMS and3,4-LICAMS are described by Raymond et al. in “Inorganic Chemistry inBiology and Medicine”, Chapter 18, ACS Symposium Series, Washington,D.C. (1980). Log stability constants are from “Critical StabilityConstants”, A. E. Martell and R. M. Smith, Vols. 1-4, Plenum Press, NY(1977); “Inorganic Chemistry in Biology and Medicine”, Chapter 17, ACSSymposium Series, Washington, D.C. (1980); R. D. Hancock and A. E.Martell, Chem. Rev. vol. 89, p. 1875-1914 (1989) and “StabilityConstants of Metal-ion Complexes”, The Chemical Society, London, 1964.

In many instances, the growth of a particular micro-organism may belimited by the availability of a particular metal-ion, for example, dueto a deficiency of this metal-ion. In such cases it is desirable toselect a metal-ion sequestrant with a very high specificity orselectivity for a given metal-ion. Metal-ion sequestrants of this naturemay be used to control the concentration of the target metal-ion andthus limit the growth of the organism(s), which require this metal-ion.However, it may be necessary to control the concentration of the targetmetal without affecting the concentrations of beneficial metal-ions suchas potassium and calcium. One skilled in the art may select a metal-ionsequestrant having a high selectivity for the target metal-ion. Theselectivity of a metal-ion sequestrant for a target metal-ion is givenby the difference between the log of the stability constant for thetarget metal-ion, and the log of the stability constant for theinterfering (beneficial) metal-ions. For example, if a treatmentrequired the removal of Fe(III), but it was necessary to leave theCa-concentration unaltered, then from Table 1, DTPA would be a suitablechoice since the difference between the log stability constants28-10.8=17.2, is very large. 3,4-LICAMS would be a still more suitablechoice since the difference between the log stability constants43-16.2=26.8, is the largest in Table 1.

It is preferred that said metal-ion sequestrant has a high-affinity foriron, and in particular iron(III). It is preferred that the stabilityconstant of the sequestrant for iron(III) be greater than 10¹⁰. It isstill further preferred that the metal-ion sequestrant has a stabilityconstant for iron greater than 10²⁰. It is still further preferred thatthe metal-ion sequestrant has a stability constant for iron greater than10³⁰.

It is preferred that the container comprises derivatized nanoparticlescomprising inorganic nanoparticles having an attached metal-ionsequestrant, wherein said inorganic nanoparticles have an averageparticle size of less than 200 nm and the derivatized nanoparticles havea stability constant greater than 10¹⁰ with iron (III). It is furtherpreferred that the derivatized nanoparticles have a stability constantgreater than 10²⁰ with iron (III). The derivatized nanoparticles arepreferred because they have very high surface area and may have a veryhigh-affinity for the target metal-ions. It is preferred that thenanoparticles have an average particle size of less than 100 nm. It isfurther preferred that the nanoparticles have an average size of lessthan 50 nm, and most preferably less than 20 nm. Preferably greater than95% by weight of the nanoparticles are less than 200 nm, more preferablyless than 100 nm, and most preferably less than 50 nm. This is preferredbecause as the particle size becomes smaller, the particles scattervisible-light less strongly. Therefore, the derivatized nanoparticlescan be applied to clear, transparent surfaces without causing a hazy ora cloudy appearance at the surface. This allows the particles of thepresent invention to be applied to packaging materials without changingthe appearance of the item. It is preferred that the nanoparticles havea very high surface area, since this provides more surface with which tocovalently bind the metal-ion sequestrant, thus improving the capacityof the derivatized nanoparticles for binding metal-ions. It is preferredthat the nanoparticles have a specific surface area of greater than 100m²/g, more preferably greater than 200 m²/g, and most preferably greaterthan 300 m²/g. For applications of the invention in which theconcentrations of contaminant or targeted metal-ions in the environmentare high, it is preferred that the nanoparticles have a particle size ofless than 20 nm and a surface area of greater than 300 m²/g. Derivatizednanoparticles are described at length in U.S. patent application Ser.No. 10/822,940 filed Apr. 13, 2004.

It is preferred that the derivatized nanoparticles have a high stabilityconstant for the target metal-ion(s). The stability constant for thederivatized nanoparticle will largely be determined by the stabilityconstant for the attached metal-ion sequestrant. However, the stabilityconstant for the derivatized nanoparticles may vary somewhat from thatof the attached metal-ion sequestrant. Generally, it is anticipated thatmetal-ion sequestrants with high stability constants will givederivatized nanoparticles with high stability constants. For aparticular application, it may be desirable to have a derivatizednanoparticle with a high selectivity for a particular metal-ion. In mostcases, the derivatized nanoparticle will have a high selectivity for aparticular metal-ion if the stability constant for that metal-ion isabout 10⁶ greater than for other ions present in the system.

Metal-ion sequestrants may be chosen from various organic molecules.Such molecules having the ability to form complexes with metal-ions areoften referred to as “chelators”, “complexing agents”, and “ligands”.Certain types of organic functional groups are known to be strong“chelators” or sequestrants of metal-ions. It is preferred that thesequestrants of the invention contain alpha-amino carboxylates,hydroxamates, or catechol, functional groups. Hydroxamates, or catechol,functional groups are preferred. Alpha-amino carboxylates have thegeneral formula:R —[N(CH₂CO₂M)-(CH₂)_(n)—N(CH₂CO₂M)₂]_(x)where R is an organic group such as an alkyl or aryl group; M is H, oran alkali or alkaline earth metal such as Na, K, Ca or Mg, or Zn; n isan integer from 1 to 6; and x is an integer from 1 to 3. Examples ofmetal-ion sequestrants containing alpha-amino carboxylate functionalgroups include ethylenediaminetetraacetic acid (EDTA),ethylenediaminetetraacetic acid disodium salt,diethylenetriaminepentaacetic acid (DTPA),Hydroxylpropylenediaminetetraacetic acid (DPTA), nitrilotriacetic acid,triethylenetetraaminehexaacetic acid, N,N-bis(o-hydroxybenzyl)ethylenediamine-N,N diacteic acid, andethylenebis-N,N′-(2-o-hydroxyphenyl)glycine.

Hydroxamates (or often called hydroxamic acids) have the generalformula:

where R is an organic group such as an alkyl or aryl group. Examples ofmetal-ion sequestrants containing hydroxamate functional groups includeacetohydroxamic acid, and desferroxamine B, the iron chelating drugdesferal.

Catechols have the general formula:

Where R1, R2, R3 and R4 may be H, an organic group such as an alkyl oraryl group, or a carboxylate or sulfonate group. Examples of metal-ionsequestrants containing catechol functional groups include catechol,disulfocatechol, dimethyl-2,3-dihydroxybenzamide, mesitylenecatecholamide (MECAM) and derivatives thereof,1,8-dihydroxynaphthalene-3,6-sulfonic acid, and2,3-dihydroxynaphthalene-6-sulfonic acid.

In a preferred embodiment the metal-ion sequestrant is attached to ananoparticle by reaction of the nanoparticle with a silicon alkoxideintermediate having the general formula:Si(OR)_(4-x)R′_(x);wherein x is an integer from 1 to 3;

-   R is an alkyl group; and R′ is an organic group containing an alpha    amino carboxylate, a hydroxamate, or a catechol. The —OR-group    attaches the silicon alkoxide to the core particle surface via a    hydrolysis reaction with the surface of the particles. Materials    suitable for practice of the invention include    N-(trimethoxysilylpropyl)ethylenediamine triacetic acid, trisodium    salt, N-(triethoxysilylpropyl)ethylenediamine triacetic acid,    trisodium salt, N-(trimethoxysilylpropyl)ethylenediamine triacetic    acid, N-(trimethoxysilylpropyl)diethylenetriamine tetra acetic acid,    N-(trimethoxysilylpropyl)amine diacetic acid, and metal-ion salts    thereof.

The antimicrobial active material of antimicrobial agent may be selectedfrom a wide range of known antibiotics and antimicrobials. Anantimicrobial material may comprise an antimicrobial ion, moleculeand/or compound, metal ion exchange materials exchanged or loaded withantimicrobial ions, molecules and/or compounds, ion exchange polymersand/or ion exchange latexes, exchanged or loaded with antimicrobialions, molecules and/or compounds. Suitable materials are discussed in“Active Packaging of Food Applications” A. L. Brody, E. R. Strupinskyand L. R. Kline, Technomic Publishing Company, Inc. Pennsylvania (2001).Examples of antimicrobial agents suitable for practice of the inventioninclude benzoic acid, sorbic acid, nisin, thymol, allicin, peroxides,imazalil, triclosan, benomyl, metal-ion release agents, metal colloids,anhydrides, and organic quaternary ammonium salts. Preferredantimicrobial reagents are metal ion exchange reagents such as silversodium zirconium phosphate, silver zeolite, or silver ion exchange resinwhich are commercially available. The antimicrobial agent may beprovided in a layer 15 having a thickness “y” of between 0.1 microns and100 microns, preferably in the range of 1.0 and 25 microns.

In another preferred embodiment, the antimicrobial agent comprising acomposition of matter comprising an immobilized metal-ionsequestrant/antimicrobial comprising a metal-ion sequestrant that has ahigh stability constant for a target metal ion and that has attachedthereto an antimicrobial metal-ion, wherein the stability constant ofthe metal-ion sequestrant for the antimicrobial metal-ion is less thanthe stability constant of the metal-ion sequestrant for the targetmetal-ion. These are explained in detail in U.S. Ser. No. 10/868,626filed Jun. 15, 2004.

In a preferred embodiment, the antimicrobial agent comprising a metalion exchange material is exchanged with at least one antimicrobial metalion selected from silver, copper, gold, nickel, tin or zinc.

Referring to FIG. 4, there is illustrated an enlarged partial crosssectional view of the wall of a fluid container 12 made in accordancewith the present invention. The wall of the container 12, which in theembodiment illustrated is a bottle, is made of a material that comprisesa barrier layer 70, an outer polymeric layer 65 and an inner polymericlayer 90 between said barrier layer 70 and outer polymeric layer 65. Theinner polymeric layer 90 contains a metal-ion sequestrant 95. Thebarrier layer 70 preferably does not contain the metal-ion sequestrant95. The outer layer 65 may provide several functions including improvingthe physical strength and toughness of the article and resistance toscratching, marring, cracking, etc. However, the primary purpose of thebarrier layer 70 is to provide a barrier through which micro-organisms80 present in the contained fluid cannot pass. It is important to limit,or eliminate, in certain applications, the direct contact ofmicro-organisms 80 with the metal-ion sequestrant 95 or the layer 90containing the metal-ion sequestrant 95, since many micro-organisms 80,under conditions of iron deficiency, may bio-synthesize molecules whichare strong chelators for iron, and other metals. These bio-syntheticmolecules are called “siderophores” and their primary purpose is toprocure iron for the micro-organisms 80. Thus, if the micro-organisms 80are allowed to directly contact the metal-ion sequestrant 95, they mayfind a rich source of iron there, and begin to colonize directly atthese surfaces. The siderophores produced by the micro-organisms maycompete with the metal-ion sequestrant for the “free” iron ion 85 (orother bio-essential metal) at their surfaces. However, the energyrequired for the organisms to adapt their metabolism to synthesize thesesiderophores will impact significantly their growth rate. Thus, oneobject of the invention is to lower growth rate of organisms in thecontained liquid. Since the barrier layer 70 of the invention does notcontain the metal-ion sequestrant 95, and because micro-organisms 80 arelarge, the micro-organisms 80 may not pass or diffuse through thebarrier layer 70. The barrier layer 70 thus prevents contact of themicro-organisms 80 with the polymeric layer 90 containing the metal-ionsequestrant 95 of the invention. It is preferred that the barrier layer70 is permeable to water. It is preferred that the barrier layer 70 hasa thickness “x” in the range of 0.1 microns to 10.0 microns. It ispreferred that microbes are unable to penetrate, to diffuse or passthrough the barrier layer 70. Sequestrant 95 with a sequesteredmetal-ion is indicated by numeral 95′.

Still referring to FIG. 4, the enlarged sectioned view of the fluidcontainer 12 shown in FIG. 2, illustrates a bottle having barrier layer70, which is in direct contact with the contained beverage 8, an innerpolymeric layer 90 and an outer polymeric layer 65. However, the priorart bottle of FIG. 3 comprises an inner polymeric layer 60 that does notcontain any metal-ion sequestering agents according to the presentinvention. In the prior art bottle illustrated in FIG. 3, themicro-organisms 80 are free to gather the “free” iron ions 85. In thebottle according to the present invention shown in FIG. 4, the innerpolymer 90 contains an immobilized metal-ion sequestering agent 95 suchas EDTA. In order for the metal-ion sequestering agent 95 to workproperly, the inner polymer 90 containing the metal-ion sequesteringagent 95 must be permeable to the aqueous solution or beverage 8.Preferred polymers for layers 70 and 90 of the invention are polyvinylalcohol, cellophane, water-based polyurethanes, polyester, nylon, highnitrile resins, polyethylene-polyvinyl alcohol copolymer, polystyrene,ethyl cellulose, cellulose acetate, cellulose nitrate, aqueous latexes,polyacrylic acid, polystyrene sulfonate, polyamide, polymethacrylate,polyethylene terephthalate, polystyrene, polyethylene, polypropylene orpolyacrylonitrile. A water permeable polymer permits water to movefreely through the polymer 90 allowing the “free” iron ion 85 to reachand be captured by the agent 95. An additional barrier 70 may be used toprevent the micro-organism 80 from reaching the inner polymer material90 containing the metal-ion sequestering agent 95 and the sequesteredmetal-ion 95′. Like the inner polymer material 90, the barrier layer 70must be made of a water permeable polymer as previously described. Themicro-organism 80 is too large to pass through the barrier 70 or theinner polymer layer 90 so it cannot reach the sequestered iron ion 95′now held by the metal-ion sequestering agent 95. By using the metal-ionsequestering agents 95 to significantly reduce the amount of “free” ironions 85 in the beverage 8, the growth of the micro-organism 80 iseliminated or severely reduced.

In the embodiment shown in FIGS. 5, 6, and 7 the metal-ion sequesteringagent 95 is contained in the bottle cap 100 instead of on the insidesurface of the bottle 12. An inner portion 105 of the cap 100, which isin intimate contact with the beverage 8, is made of a hydrophilicpolymer 110 containing the metal-ion sequestering agent 95 such as EDTAas described above. In some situations, the bottle 8 may need to beplaced in the inverted position in order for the sequestrant to come incontact with the contained nutrient. The cap 100 may also have thebarrier layer 70 to further prevent the micro-organisms 80 from reachingthe sequestered “free” iron ion 95′. In another embodiment (not shown)the cap sealing material could be an open cell foamed structure whosecell walls are coated with the sequestering material.

In another embodiment of the present invention, the sequestering agent95 may be in a hydrophilic polymeric insert 115 that is placed in thebottle 12 as illustrated in FIG. 5. The insert 115 may be instead of orin addition to the sequestrant in the cap 100 or interior of the bottle.The insert 115 is placed in the bottle 12 but unfolds making it toolarge to exit the bottle 12. In another version, the insert 115 ismolded into the bottom of the bottle 12.

Referring to FIGS. 8 and 9, there is illustrated yet another embodimentof a bottle 12 made in accordance with the present invention. In thisembodiment, the metal-ion sequestering agent 95 is applied to theinterior surface 120 of the bottle 12 by spraying a metal-ionsequestering agent 95, for example EDTA, on to the interior surface 120of the bottle 12, through a supply tube 125 using a spherical shapednozzle assembly 130. The nozzle assembly 130 is moved up and down in thedirection of the arrow 135 while the metal-ion sequestering agent 95 issprayed as indicated by the arrows 140. It is to be understood that anymethod of applying coatings to glass, metal or plastic containers may beused as is well known to those skilled in the art of applying suchcoating. FIG. 9 illustrates an enlarged partial cross sectional view ofthe portion of the bottle of FIG. 8 where the spray coating 150 of themetal-ion sequestering agent 95 has been applied. As previouslydiscussed in FIG. 4, like numerals indicate like parts and operations.It is of course understood that the inner layer containing thesequestrant may be applied or formed on the inside surface of thecontainer in any appropriate manner. The bottle 12 in this embodimentmay be made of any appropriate plastic or glass material. While in theembodiment illustrated substantially the entire interior surface 120 iscoated with the metal-ion sequestering agent 95, the present inventionis not so limited. Only that portion of the interior surface need becoated as necessary for requesting the desired free metal-ion and anyappropriate pattern.

By using the metal-ion sequestering agents 95 to remove “free” iron 85as the method for eliminating the micro-organisms 80 that enter thebottles 12 between the filling station 25 and the capper 30, the “hotfill” portion 40 of the process shown in FIG. 1 is no longer necessary.The use of bottles 200, bags, stand up pouches, juice boxes, cans, etccontaining metal-ion sequestering agents 95 as described in FIGS. 4through 9, the process 205 shown in FIG. 10 may be used for bottling thetypes of beverages and foodstuffs requiring the “hot fill” process. Theprocess of FIG. 10 is similar to that of FIG. 1, like numbers indicatelike parts and operations as previously discussed, except that theheating tunnel 40 where the beverage is heated and the cooling tunnel 45for cooling of the heated bottles are eliminated as they are not neededor is significantly reduced. By removing and/or significantly reducingthe “hot fill” portion 40 (shown in FIG. 1) of the process 205, theamount of energy required for both heating and cooling the bottlesduring the filling process is greatly reduced while increasing theoptions in both bottle design and materials to be used in the bottlingprocess. Depending on the liquid being bottled and the iron-sequesteringagent and/or antimicrobial agent being utilized, some heating andcooling may be required, but at significantly reduced level whereby adirect a significantly economic benefit will be realized. For example, asaving as little as about one cent ($0.01) may be realized, this wouldbe very significant as a typically bottling plant will fill millions ofbottles per year. It is of course understood that the certain processesof FIG. 10 may be further modified or eliminated depending on the typeof container being used. For example, where a drink box, drink bag, can,is used, a different type of filler or capping/closure device may beutilized as required.

Examples and Comparison Examples

Materials:

Colloidal dispersions of silica particles were obtained from ONDEO NalcoChemical Company. NALCO® 1130 had a median particle size of 8 nm, a pHof 10.0, a specific gravity of 1.21 g/ml, a surface area of about 375m²/g, and a solids content of 30 weight.N-(trimethoxysilylpropylethylenediamine triacetic acid, trisodium saltwas purchased from Gelest Inc., 45% by weight in water.

Preparation of derivatized nanoparticles. To 600.00 g of silica NALCO®1130 (30% solids) was added 400.00 g of distilled water and the contentsmixed thoroughly using a mechanical mixer. To this suspension, was added49.4 g of N-(trimethoxysilyl)propylethylenediamine triacetic acid,trisodium salt in 49.4 g distilled water with constant stirring at arate of 5.00 ml/min. At the end of the addition the pH was adjusted to7.1 with the slow addition of 13.8 g of concentrated nitric acid, andthe contents stirred for an hour at room temperature. Particle sizeanalysis indicated an average particle size of 15 nm. The percent solidsof the final dispersion was 18.0%.

Preparation of the immobilized metal-ion sequestrant/antimicrobial:200.0 g of the above derivatized nanoparticles were washed withdistilled water via dialysis using a 6,000-8,000 molecular weight cutofffilter. The final ionic strength of the solution was less than 0.1millisemens. To the washed suspension was then added with stirring 4.54ml of 1.5 M AgNO₃ solution, to form the immobilized metal-ionsequestrant/antimicrobial.

Preparation of Polymeric Layers of Immobilized Metal-Ion Sequestrantsand Sequestrant/Antimicrobials.

Coating 1 (comparison). A coating solution was prepared as follows: 8.8g of a 40% solution of the polyurethane Permax 220 (Noveon Chemicals)was combined with to 90.2 grams of pure distilled water and 1.0 g of a10% solution of the surfactant OLIN 10G was added as a coating aid. Themixture was then stirred and blade-coated onto a polymeric support usinga 150 micron doctor blade. The coating was then dried at 40-50° C., toproduce a film having 5.4 g/m² of polyurethane.

Coating 2. A coating solution was prepared as follows: 171.2 grams ofthe derivatized nanoparticles prepared as described above were combinedwith 64.8 grams of pure distilled water and 62.5 g of a 40% solution ofthe polyurethane Permax 220 (Noveon Chemicals). 1.5 g of a 10% solutionof the surfactant OLIN 10G was added as a coating aid. The mixture wasthen stirred and blade-coated onto a polymeric support using a 150micron doctor blade. The coating was then dried at 40-50° C., to producea film having 5.4 g/m² of the derivatized nanoparticles and 5.4 g/m² ofpolyurethane.

Coating 3. A coating solution was prepared as follows: 171.2 grams ofthe derivatized nanoparticles prepared as described above were combinedwith 33.5 grams of pure distilled water and 93.8 g of a 40% solution ofthe polyurethane Permax 220 (Noveon Chemicals). 1.5 g of a 10% solutionof the surfactant OLIN 10G was added as a coating aid. The mixture wasthen stirred and blade-coated onto a polymeric support using a 150micron doctor blade. The coating was then dried at 40-50° C., to producea film having 5.4 g/m² of the derivatized nanoparticles and 8.1 g/m² ofpolyurethane.

Coating 4. A coating solution was prepared as follows: 138.9 grams ofthe derivatized nanoparticles prepared as described above were combinedwith 97.1 grams of pure distilled water and 62.5 g of a 40% solution ofthe polyurethane Permax 220 (Noveon Chemicals). 1.5 g of a 10% solutionof the surfactant OLIN 10G was added as a coating aid. The mixture wasthen stirred and blade-coated onto a polymeric support using a 150micron doctor blade. The coating was then dried at 40-50° C., to producea film having 4.4 g/m² of the derivatized nanoparticles and 5.4 g/m² ofpolyurethane.

Coating 5. A coating solution was prepared as follows: 12.8 grams of theimmobilized metal-ion sequestrant/antimicrobial suspension prepared asdescribed above was combined with to 77.4 grams of pure distilled waterand 8.8 g of a 40% solution of the polyurethane Permax 220 (NoveonChemicals). 1.0 g of a 10% solution of the surfactant OLIN 10G was addedas a coating aid. The mixture was then stirred and blade-coated onto apolymeric support using a 150 micron doctor blade. The coating was thendried at 40-50° C., to produce a film having 2.7 g/m² of the immobilizedmetal-ion sequestrant/antimicrobial, 0.06 g/m² silver-ion and 5.4 g/m²of polyurethane.

Testing Methodology

A test similar to ASTM E 2108-01 was conducted where a piece of acoating of known surface area was contacted with a solution inoculatedwith micro-organisms. In particular, a piece of coating 1×1 cm wasdipped in 2 ml of growth medium (Trypcase Soy Agar 1/10), inoculatedwith 2000 CFU of Candida albicans (ATCC-1023) per ml. Special attentionwas made to all reagents to avoid iron contamination with the finalsolution having an iron concentration of 80 ppb before contact with thecoating.

Micro-organism numbers in the solution were measured daily by thestandard heterotrophic plate count method.

BAR GRAPH 1 demonstrates the effectiveness of the inventive examples.The yeast population which was exposed to the comparison coating 1(which contained no derivatized nanoparticles) showed a growth factor ofone thousand during 48 hours (a 1000-fold increase in population). Theyeast population which was exposed to the example coatings 2-4(containing derivatized nanoparticles) showed growth factors of only1-4. This is indicative of a fungostatic effect in which the populationof organisms is kept at a constant or near constant level, even in thepresence of a medium containing adequate nutrient level. The yeastpopulation which was exposed to the example coating 5 (derivatizednanoparticles that had been ion exchanged with silver ion—a knownantimicrobial) showed a fungicidal effect (the yeast were completelyeliminated). The low level of silver when coated by itself without thenanoparticles would not be expected to exhibit this complete fungicidaleffect, and there appears to be a synergistic effect between the ironsequestration and the release of antimicrobial silver.

As can be seen from BAR GRAPH 1, significant improved results may beobtained when a metal-ion sequestering agent is used in conjunction withan antimicrobial agent. The combined agents reduced the level ofmicrobes to lower level than when first introduced and then maintainedthe reduced level of microbes in the liquid nutrient.

The invention has been described in detail with particular reference tocertain preferred embodiments thereof, but it will be understood thatvariations and modifications can be effected within the scope of theinvention, the present invention being defined by the claim set forthherein.

Parts List

-   5 process-   8 beverage-   10 tank-   12 container/bottle-   15 pump-   20 pasteurizer-   25 filler station-   26 line-   27 processing path-   30 barrier layer-   35 capper-   40 heating tunnel-   45 cooling tunnel-   50 packing station-   55 shipping station-   60 inner polymer material-   65 outer polymer material-   70 barrier layer-   80 micro-organism-   85 “free” iron ion-   90 inner polymer-   95 metal-ion sequestering agent-   95′ sequestered metal-ion-   100 cap-   105 inner portion-   110 hydrophilic layer-   115 insert-   120 inside surface-   125 supply tube-   130 nozzle assembly-   135 arrow-   140 arrow-   150 spray coating-   200 process-   205 bottle

1. A method of removing a selected metal-ion from a solution, comprisingthe steps of: a. providing a container for holding a liquid, saidcontainer having an internal surface having a metal-ion sequesteringagent provided on at least a portion of said internal surface forremoving a designated metal-ions from said liquid and an antimicrobialagent for reducing and/or maintaining the amount of microbes in saidliquid to a prescribed condition; b. filling said container with saidliquid in an open environment; c. closing said container with saidliquid contained therein; and d. shipping said container for use of saidliquid without any further processing of said container containing saidliquid.
 2. A method according to claim 1 wherein said container ispositioned such that said metal-ion sequestering agent and saidantimicrobial agent contacts said liquid for a time period sufficientfor removing said designated metal-ions from said liquid and forreducing and/or maintaining the amount of microbes in said liquid to aprescribed condition.
 3. A method according to claim 2 wherein saidcontainer comprises a bottle and cap assembly.
 4. A method according toclaim 3 wherein said bottle is made of a plastic material.
 5. A methodaccording to claim 3 wherein said metal-ion sequestering agent and/orantimicrobial agent is provided on the internal surface of said bottle.6. A method according to claim 3 wherein said bottle is made of amaterial that includes said metal-ion sequestering agent and/orantimicrobial agent.
 7. A method according to claim 1 wherein saidmetal-ion sequestering agent and/or said antimicrobial agent is providedon the internal surface of said cap.
 8. A method according to claim 1wherein said liquid has a pH equal to or greater than about
 3. 9. Amethod according to claim 1 wherein said antimicrobial agent comprisesan antimicrobial active material selected from benzoic acid, sorbicacid, nisin, thymol, allicin, peroxides, imazalil, triclosan, benomyl,metal-ion release agents, metal colloids, anhydrides, and organicquaternary ammonium salts, a metal ion exchange reagents such as silversodium zirconium phosphate, silver zeolite, or silver ion exchangeresin.
 10. A method according to claim 1 wherein said metal-ionsequestering agent is immobilized on the surface(s) of said containerand has a stability constant greater than 10¹⁰ with iron (III).
 11. Amethod according to claim 1 wherein said sequestering agent isimmobilized on the surface(s) of said container and has a high-affinityfor biologically important metal-ions such as Mn, Zn, Cu and Fe.
 12. Amethod according to claim 1 wherein said antimicrobial agent comprises ametal ion selected from one of the following: silver; copper; gold;nickel; tin; zinc.
 13. A method according to claim 1 wherein saidsequestering agent has a high-selectively for certain metal-ions but alow-affinity for at least one other ion.
 14. A method according to claim1 wherein said metal-ion sequestering agent is immobilized on thesurface(s) of said container and has a stability constant greater than10¹⁰ with iron (III) and said antimicrobial agent comprises anantimicrobial active material selected from benzoic acid, sorbic acid,nisin, thymol, allicin, peroxides, imazalil, triclosan, benomyl,metal-ion release agents, metal colloids, anhydrides, and organicquaternary ammonium salts. Preferred antimicrobial reagents are metalion exchange reagents such as silver sodium zirconium phosphate, silverzeolite, or silver ion exchange resin.
 15. A method according to claim 1wherein said metal-ion sequestering agent is immobilized on thesurface(s) of said container and has a stability constant greater than10²⁰ with iron (III).
 16. A method according to claim 1 wherein saidmetal-ion sequestering agent is immobilized on the surface(s) of saidcontainer and has a stability constant greater than 10¹⁰ with iron (III)and said antimicrobial agent comprises a metal ion selected from one ofthe following: silver; copper; gold; nickel; tin; zinc.
 17. A methodaccording to claim 1 wherein said metal-ion sequestering agent comprisesderivatized nanoparticles comprising inorganic nanoparticles having anattached metal-ion sequestrant, wherein said inorganic nanoparticleshave an average particle size of less than 200 nm and the derivatizednanoparticles have a stability constant greater than 10¹⁰ with iron(III).
 18. A method according to claim 1 wherein said metal-ionsequestering agent is immobilized in a polymeric layer, and thepolymeric layer contacts the fluid contained therein.
 19. A methodaccording to claim 1 wherein said antimicrobial agent maintains saidmicrobes in a biostatic state.
 20. A method according to claim 1 whereinsaid antimicrobial agent maintains said microbes in a substantiallybiocide state.
 21. A method according to claim 1 wherein saidantimicrobial agent maintains said microbes to a prescribed level.
 22. Amethod according to claim 1 wherein said antimicrobial agent maintainssaid microbes to a level that will not harm users.
 23. A method forbottling a liquid having a pH equal to or greater than about 2.5,comprising the steps of: a. providing a container having a metal-ionsequestering agent and an antimicrobial agent provided on at least aportion of said internal surface for inhibiting growth of microbes; b.filling said container with a liquid having a pH equal to or greaterthan about 2.5; c. closing said container with said liquid containedtherein; and d. shipping said container for use without any furthersterilization of said liquid and/or container.
 24. A method according toclaim 23 wherein said container comprises a bottle and cap.
 25. A methodaccording to claim 23 wherein metal-ion sequestering agent and/or saidantimicrobial agent is provided on the interior surface of said bottle.26. A method according to claim 23 wherein metal-ion sequestering agentand/or said antimicrobial agent is provided on the interior surface ofsaid cap.
 27. A method according to claim 23 wherein said bottle is madeof a material that includes said metal-ion sequestering agent.
 28. Amethod according to claim 23 wherein said liquid is a beverage that isconsumed by individuals.
 29. A method according to claim 23 wherein saidpH is equal to or greater than 3.0.
 30. A method according to claim 23wherein said pH is equal to or greater than 4.0.
 31. An article forinhibiting the growth of microbes in a liquid nutrient when placed incontact with the liquid nutrient, said article having a metal-ionsequestering agent and an antimicrobial agent such that when saidarticle is placed in contact with said liquid nutrient said metal-ionsequestering agent and said antimicrobial agent inhibits the growth ofmicrobes in said liquid nutrient.
 32. An article according to claim 31wherein said metal-ion sequestering agent and/or antimicrobial agent issecured to said article by a support structure.
 33. An article accordingto claim 31 wherein said metal-ion sequestering agent is immobilized onthe surface(s) of said container and has a stability constant greaterthan 10¹⁰ with iron (III).
 34. An article according to claim 31 whereinsaid sequestering agent is immobilized on the surface(s) of saidcontainer and has a high-affinity for biologically important metal-ionssuch as Mn, Zn, Cu and Fe.
 35. An article according to claim 31 whereinsaid sequestering agent is immobilized on the surface(s) of saidcontainer and has a high-selectivity for biologically importantmetal-ions such as Mn, Zn, Cu and Fe.
 36. An article according to claim31 wherein said sequestering agent has a high-selectively for certainmetal-ions but a low-affinity for at least one other ion.
 37. An articleaccording to claim 36 wherein said certain metal-ions comprises Mn, Zn,Cu and Fe and said other at least one ion comprises calcium.
 38. Anarticle according to claim 31 wherein said metal-ion sequestering agentis immobilized on the surface(s) of said container and has a stabilityconstant greater than 10¹⁰ with iron (III) and said antimicrobial agentcomprises an antimicrobial active material selected from benzoic acid,sorbic acid, nisin, thymol, allicin, peroxides, imazalil, triclosan,benomyl, metal-ion release agents, metal colloids, anhydrides, andorganic quaternary ammonium salts. Preferred antimicrobial reagents aremetal ion exchange reagents such as silver sodium zirconium phosphate,silver zeolite, or silver ion exchange resin.
 39. An article accordingto claim 31 wherein said metal-ion sequestering agent is immobilized onthe surface(s) of said container and has a stability constant greaterthan 10³⁰ with iron (III).
 40. An article according to claim 31 whereinsaid metal-ion sequestering agent comprises derivatized nanoparticlescomprising inorganic nanoparticles having an attached metal-ionsequestrant, wherein said inorganic nanoparticles have an averageparticle size of less than 200 nm and the derivatized nanoparticles havea stability constant greater than 10¹⁰ with iron (III).
 41. An articleaccording to claim 31 wherein said metal-ion sequestering agent and/orantimicrobial agent is immobilized in a polymeric layer, and thepolymeric layer contacts the fluid contained therein.
 42. An articleaccording to claim 31 wherein said antimicrobial agent maintains saidmicrobes in a biostatic state.
 43. An article according to claim 31wherein said antimicrobial agent maintains said microbes in asubstantially biocide state.
 44. An article according to claim 31wherein said antimicrobial agent maintains said microbes to a prescribedlevel.
 45. An article according to claim 31 wherein said antimicrobialagent maintains said microbes to a level that will not harm users ofsaid article
 46. A method of removing a selected metal-ion from asolution, comprising the steps of: a. providing a container for holdinga liquid, said container having an internal surface having a metal-ionsequestering agent provided on at least a portion of said internalsurface for removing a designated metal-ion from said liquid forreducing and/or maintaining the amount of microbes in said liquid to aprescribed condition wherein the heating or cooling of said containerafter filling is substantially reduced; b. filling said container withsaid liquid in an open environment; c. closing said container with saidliquid contained therein; d. heating of said filled closed container; e.cooling of said heated container; and f. shipping said container for useof said liquid without any further processing of said containercontaining said liquid.
 47. A method for bottling a liquid having a pHequal to or greater than about 2.5, comprising the steps of: a.providing a container having a metal-ion sequestering agent on at leasta portion of said internal surface for inhibiting growth of microbeswherein the heating or cooling of said container after filling issubstantially reduced; b. filling said container with a liquid having apH equal to or greater than about 2.5; c. closing said container withsaid liquid contained therein; d. heating of said filled closedcontainer; e. cooling of said heated container; and f. shipping saidcontainer for use without any further sterilization of said liquidand/or container.